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目的制备针对胚胎小鼠心肌L型钙通道Cav1.3编码的α1D亚基胞内末端的多克隆抗体。方法通过基因重组从小鼠胚胎心脏中获得2种原核表达质粒pGEX-4T-α1DN/α1DC,在大肠杆菌中经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达谷胱甘肽S转移酶(GST)融合蛋白,纯化得到α1D基因N端和C端产物GST-α1DN/α1DC,用此抗原经弗式佐剂、弗式不完全佐剂乳化后免疫新西兰家兔,收集的免疫血清经protein G Agarose亲和纯化后获得IgG型抗α1D蛋白的多克隆抗体。对这2种抗体采用Westernblot法进行特异性检测,ELISA法测定进行效价分析。结果成功构建α1D胞内片段的原核表达载体,表达纯化了GST融合蛋白GST-α1DC/α1DN,制备2种IgG型多克隆抗体,经ELISA检验证实二者效价均较高,滴度在1:256000时仍有信号,其中GST-α1DN的效价更高。用Westernblot法检测发现,2种抗体都均在相对分子质量240000处得到单一的蛋白印迹条带,特异性良好,能识别小鼠心肌细胞中α1D蛋白。结论用重组融合蛋白GST-α1D作为抗原制备出了具有高效价、强特异性的α1D抗体,为探讨α1D蛋白在小鼠胚胎心脏发育过程中分布的时空特征及研究胚胎发育过程中的钙离子相关信号调控活动奠定了基础。
Objective To prepare polyclonal antibodies targeting the intracellular end of α1D subunit encoded by L type calcium channel Cav1.3 in embryonic mice. Methods Two prokaryotic expression plasmids, pGEX-4T-α1DN / α1DC, were obtained from mouse embryonic heart by gene recombination. Glutathione S was induced by isopropyl-β-D thiogalactoside (IPTG) in Escherichia coli Transfected enzyme (GST) fusion protein was purified to obtain the N-terminal and C-terminal GST-α1DN / α1DC of the α1D gene. Immune New Zealand rabbits were immunized with this antigen by means of an adjuvant and an incomplete Freund’s adjuvant. The collected immune serum After affinity G protein purification, the IgG anti-α1D polyclonal antibody was obtained. The specific antibodies against these two antibodies were detected by Western blot and the titer analysis by ELISA. Results The prokaryotic expression vector of α1D intracellular fragment was successfully constructed and the GST fusion protein GST-α1DC / α1DN was expressed and purified. Two kinds of IgG polyclonal antibodies were prepared. The titer of the two polyclonal antibodies against IgG was higher than 1: There is still a signal at 256000, where the titer of GST-α1DN is higher. Western blot analysis showed that both antibodies were single-stranded with a molecular weight of 240,000, which showed good specificity and could recognize α1D protein in mouse cardiomyocytes. Conclusion The recombinant fusion protein GST-α1D was used as an antigen to prepare high titer and strong specificity α1D antibody. To investigate the spatial and temporal distribution of α1D protein in mouse embryonic heart development and to study the correlation between calcium and calcium during embryonic development Signal conditioning activities laid the foundation.